PROJECT SUMMARY Regulated exocytosis ? stimulus-dependent exocytic vesicle fusion ? mediates a broad range of fundamental biological processes including nutrient homeostasis, hormonal signaling, synaptic transmission, and elimination of transformed or virally infected cells. Imbalances in these exocytic pathways lead to major forms of human disease such as diabetes, neurological disorders, immunodeficiency, and cancer. The overall goal of this research is to establish the molecular principles of regulate exocytosis, using the trafficking of the glucose transporter GLUT4 as a model system. In our previous research, we delineated the molecular mechanisms of known exocytic regulators in the GLUT4 pathway. In this research, we will focus on a group of new regulatory factors identified in our recent genome-wide CRISPR screens investigating GLUT4 exocytosis. We will carry out in-depth biochemical, biophysical, cell biological, and genetic studies to address two key questions: 1) How do exocytic mediators act in concert to drive exocytic vesicle fusion? 2) How is exocytic vesicle fusion coupled to other cellular processes to achieve an integrated response? To answer the first question, we will define the molecular mechanisms by which SNARE-binding regulators, alone and in combination, control SNARE zippering, membrane tethering, bilayer curvature generation, lipid mixing, and content mixing. To answer the second question, we will determine whether and how the GLUT4 exocytic pathway is influenced by stimulus- dependent phosphorylations on SNAREs, conserved SM proteins, specialized exocytic regulators, and mediators of clathrin-mediated endocytosis. We will also investigate the functional roles and molecular basis of cargo retrieval in establishing an integrated exocytic response. Besides these mechanistic analyses, we will continue to identify, validate and characterize new trafficking regulators based on the candidate genes from our unbiased and targeted CRISPR screens. The mechanistic studies and CRISPR screens are fully complementary and will provide a comprehensive understanding of exocytosis regulation that neither approach alone could generate. Successful completion of this proposed research will fill major gaps in the knowledge of regulated exocytosis and will serve as a springboard for understanding the general principles of membrane trafficking. Ultimately, insights gleaned from this work will facilitate the development of new therapeutic strategies for diseases caused by dysregulated exocytosis.